Intestinal stem cells (ISCs) are critical drivers of epithelial homeostasis and regeneration. There are major gaps in knowledge concerning the identities of distinct ISC populations and the roles of epigenetic regulators in ISC self-renewal and multipotency. Lack of such knowledge has significantly hindered the understanding of the functions of ISCs, as well as the regulatory networks involved, in intestinal homeostasis and regeneration. The long-term goal of the applicant's research is to harness the reversibility of epigenetic modifications, including post-translational modifications of histones, to promote tissue regeneration and repair. The objective of this application is to determine the role of Eset (also known as Setdb1 and KMT1E), a histone H3K9 methyltransferase expressed in a fraction of ISCs, in ISC functions such as intestinal homeostasis and regeneration. The central hypothesis is that Eset specifically marks active ISCs and acts as a key corepressor of transcription factors in regulating a gene program that is essential for the maintenance or functioning of these cells. This hypothesis has been formulated on the basis of preliminary data generated in the applicant's laboratory. The rationale for the proposed research is that defining the identity and functions of active ISCs and understanding how they are regulated have the potential to deliver preventive and therapeutic value for intestinal damage, which is frequently associated with chemo- and radiotherapy, drug-mediated toxicity, and various inflammatory disorders. Guided by strong preliminary data, this hypothesis will be tested in mouse models by pursuing three specific aims: 1) Validate Eset as a specific marker of active intestinal stem cells; 2) Determine the role of Eset in intestinal stem cell functions; and 3) Elucidate the molecular mechanisms by which Eset exerts its function in intestinal stem cells. Specifically, the applicant proposes to compare the proliferation rates of Eset-positive and -negative ISCs and identify other key properties (radiosensitivity, transcriptional profile) of Eset-positive ISCs (aim 1), to determine the effects of Eset deficiency and overexpression on intestinal homeostasis and regeneration in vivo and on ISC viability, self-renewal, and differentiation ex vivo (aim 2), and to identify Eset upstream regulators and downstream effectors and to elucidate the mechanistic interactions between Eset and key transcription factors in the regulation of gene expression in ISCs (aim 3). The proposed research is innovative, in the applicant's opinion, because the concept that Eset specifically marks active ISCs is novel and innovative approaches (e.g. in vivo lineage tracing, ex vivo ISC culture, histone peptide arrays) will be used. The project is significant, because results from the proposed studies are expected to vertically advance the fields of intestinal stem cells and epithelial biology by further defining the identity and functions of active ISCs and providing novel insights into epigenetic regulation of ISC functions. These results also have potential translational impact. The restricted expression of Eset in ISCs makes it a promising target for preventive and therapeutic interventions for intestinal damage.